EP0126009B1 - Peptide derivatives, their preparation and use as elastase inhibitors - Google Patents

Abstract

Lipopeptides of the formula, R-X-(P1)x-(L-Ala-L-Ala-P2-A wherein x is 0 or 1, R is the acyl residue of a hydrophobic carboxylic acid, P2 is, for instance, L-Ala, L-Val, L-Pro-L-Ala or L-Pro-L-Val, P1 is a residue of an amino acid or an oligopeptide formed from 2-8 amino acids, X represent a divalent group or a direct covalent bond and A represents the C-terminal portion (modified or not) of -(P1)x-(Ala-Ala-P2). These lipopeptides can be used as elastase inhibitors.

Description

Elastin is known to be an elastic fibrous protein in the connective tissue of vertebrates. It is present in the vascular walls, skin, lungs, cartilage, ligaments and other tissues. Elastin is the most resistant protein in the body. On the other hand, its degradation increases particularly quickly in certain pathological states and in general during aging, in all tissues rich in elastin such as the vascular walls and the dermis; see L. ROBERT, in "Précis de physiologie cutané", edited by J. MEYNADIER, Ed. de la Porte Verte (1980) p. 155-173.

Only a few proteases can attack elastin. These proteases are called elastases or proteases of the elastase type. Such enzymes are pancreatic elastase and cellular elastases: leukocyte and platelet elastases, and elastases of macrophages, fibroblasts and arterial smooth muscle cells.

These enzymes are capable of degrading elastin in the tissues and organs mentioned above, and contributing to the development of diseases such as arteriosclerosis, emphysema, osteoarthritis, diabetes, and also to the aging of connective tissues of the organism.

The activity of the elastases is controlled and regulated by natural inhibitors which are present on the one hand in the blood plasma, such as alpha 1-antitrypsin and alpha 2-macroglobulin and on the other hand in tissue secretions such as bronchial secretions; see for example HORNEBECK et al: "Control of elastic tissue destruction by elastase inhibitors" in DEYL, ADAM Eds, Connective Tissue Research: Chemistry, Biology and Physiology, p. 233-246, A.R. Liss. Inc., New York 1981.

In addition, many bacteria capable of entering the body also secrete elastolytic proteases, the action of which contributes substantially to their pathogenic effect.

We also know that the progression of malignant tumors (cancers, sarcomas) in the body and the formation of metastases, often fatal for the patient, are also conditioned by the secretion of proteases of the elastage type; see for example Biological significance of Elastase-like enzymes in Arteriosclerosis and Human breast cancer. HORNEBECK W., BRECHEMIER D., BELLON C., ADNET J.J. and ROBERT L. in: P. Straûlli, A.J. Barrett, A. Baici eds. Proteinases and tumor invasion. vol 6, of ORTC Monograph series (1980) pp. 117-141, (Raven Press, New York). Such enzymes are capable of destroying the surrounding tissue and thus making it possible for malignant cells to enter the bloodstream and cause the tumor to invade the body.

For all these reasons, it is important to be able to have inhibitors capable of controlling the activity of elastases.

However, some of these elastases have a useful, even essential, activity for the organism, for example in the digestion of bacteria phagocytosed by macrophages.

It therefore appears important to have both elastase inhibitors and elastic fiber protectors. It also seems preferable to be able to have inhibitors of elastases which are capable of acting selectively at the level of the elastic fibers whose integrity is essential for the proper functioning of the organism.

Indeed, the enzymatic hydrolysis of elastin by elastases can be considered as a determining factor in many pathologies of elastic tissues: iberiosclerosis, emphysema and certain skin diseases. In the living organism, this proteolysis results from an imbalance between the level of proteases having an elastolytic activity on the one hand and on the other hand the rate of natural inhibitors of plasma or tissue origin. One of the therapeutic approaches which has been considered in the case of a deficiency of genetic or functional origin of these protease inhibitors, consists in the use of natural substitution inhibitors (alpha 1-antitrypsin).

However, the use of natural inhibitors has many disadvantages, including the cost of treatment and the risk of accidents of immunological origin. On the other hand, the elastase inhibitors used in experimental animal therapy in the case of emphysema have the drawback of having a high toxicity.

The present invention relates to novel bifunctional synthetic lipopeptides which can be considered both as inhibitors of elastolytic activity and protectors of elastic fiber. These substances are in fact capable of recognizing and fixing the elastic fiber and on the other hand of recognizing and neutralizing the active site of the elastases.

The elastase inhibitors of the invention have in particular the following advantages: they do not have an antigenic character; they are biodegradable; they also have the property of reaching and settling on the site of their action.

• R-7C-(_P ^yX (L-Ala-L-Ala-Pz)-A (I)

dans laquelle:

• x représente le nombre 0 ou 1;
• R représente le reste acyle d'un acide carboxylique hydrophobe tel qu'un acide carboxylique aliphatique ayant 6 à 25 atomes de carbone et pouvant renfermer éventuellement 1 à 5 doubles liaisons, d'un acide carboxylique alicyclique ayant 6 à 25 atomes de carbone, d'un acide arylcarboxylique ou d'un acide carboxylique arylaliphatique dont le groupement aryle comprend 1 à 2 cycles et dont le groupement aliphitique comprend 1 à 18 atomes de carbone, les groupements aromatiques desdits groupements aryle ou arylaliphatique étant éventuellement substitués;
• P₂ représente un résidu d'acide aminé ou de dipeptide, relié par son extrémité N-terminale au groupement L-Ala adjacent, choisi dans le groupe constitué par les acides aminés et dipeptides suivants:
  • -L-Ala-, -L-Val-, -Gly-, -L-Mét-, -L-Pro-, -L-Leu-, -L-Pro-L-Val-, -L-Pro-L-Ala-, -L-Pro-L-Phé-, -L-Pro-L-Leu, -L-Pro-L-Mét- et -L-Pro-Gly-;
• P₁ représente un résidu d'acide aminé ou d'oligopeptide formé de 2 à 8 aminoacides de la série L, lesdits aminoacides étant choisis dans le groupe constitué par la glycine, l'alanine, la valine, la méthionine, la leucine, l'isoleucine, la proline, la phénylalanine, la sérine, la cystéine, la cystine, l'arginine, la tyrosine, l'ornithine, la lysine et l'acide glutamique,
• étant entendu que P₁ est relié à RX- par son groupement N-terminal et que P₂ est relié à A par son groupement C-terminal,
• Ala est la représentation conventionnelle de l'alanine,
• X représente une liaison covalente directe reliant R soit au groupement N-terminal (-NH-) du premier aminoacide de P₁ (cas où x = 1) soit au groupement N-terminal (-NH-) du premier groupement Ala représenté à gauche sur la formule 1 (cas où x = 0),
• ou bien X est un groupement divalent ayant 2 à 10 atomes de carbone jouant le rôle de "bras" entre le groupement R et le reste de la molécule de formule I,
• et A représente la partie C-terminale du peptide -(P₁)_x-(Ala-Ali-P₂) -A, A étant choisi parmi un groupement carboxylique -C0₂H ou ses dérivés, -CHO, -CONH₂, -COCH₂CI et -CH₂0H.

The present invention therefore relates to new lipopeptides of general formula:

• R-7C- ( _P ^y X (L-Ala-L-Ala-Pz) -A (I)

in which:

• x represents the number 0 or 1;
• R represents the acyl residue of a hydrophobic carboxylic acid such as an aliphatic carboxylic acid having 6 to 25 carbon atoms and possibly containing 1 to 5 double bonds, of an alicyclic carboxylic acid having 6 to 25 carbon atoms, d '' an arylcarboxylic acid or an arylaliphatic carboxylic acid whose aryl group comprises 1 to 2 rings and whose aliphitic group comprises 1 to 18 carbon atoms, the aromatic groups of said aryl groups or arylaliphatic being optionally substituted;
• P ₂ represents an amino acid or dipeptide residue, linked by its N-terminal end to the adjacent L-Ala group, chosen from the group consisting of the following amino acids and dipeptides:
  • -L-Ala-, -L-Val-, -Gly-, -L-Mét-, -L-Pro-, -L-Leu-, -L-Pro-L-Val-, -L-Pro-L -Ala-, -L-Pro-L-Phe-, -L-Pro-L-Leu, -L-Pro-L-Met- and -L-Pro-Gly-;
• P ₁ represents an amino acid or oligopeptide residue formed from 2 to 8 amino acids of the L series, said amino acids being chosen from the group consisting of glycine, alanine, valine, methionine, leucine, l isoleucine, proline, phenylalanine, serine, cysteine, cystine, arginine, tyrosine, ornithine, lysine and glutamic acid,
• it being understood that P ₁ is linked to RX- by its N-terminal grouping and that P ₂ is linked to A by its C-terminal grouping,
• Ala is the conventional representation of alanine,
• X represents a direct covalent bond connecting R either to the N-terminal group (-NH-) of the first amino acid of P ₁ (case where x = 1) or to the N-terminal group (-NH-) of the first Ala group represented on the left on formula 1 (case where x = 0),
• or X is a divalent group having 2 to 10 carbon atoms playing the role of "arm" between the group R and the rest of the molecule of formula I,
• and A represents the C-terminal part of the peptide - (P ₁ ) _x - (Ala-Ali-P ₂ ) -A, A being chosen from a carboxylic group -C0 ₂ H or its derivatives, -CHO, -CONH ₂ , -COCH ₂ CI and -CH ₂ 0H.

Among the derivatives of formula 1, mention will be made in particular of those for which R represents the acyl residue of a fatty acid having 6 to 20 carbon atoms such as lauric acid or oleic acid or the remainder of another organic acid with hydrophobic character such as the rest of chenodeoxycholic acid, cholic acid, etc ...; or R- represents, for example, the acyl residue of a phenylalkanoic acid optionally substituted on the benzene ring (for example by halogen, triflourmethyl, hydroxyl or lower alkyl groups having 1 to 3 carbon atoms); when X is a divalent group playing the role of arm it is for example a group -Z- (CH ₂ ) _n -CO-, Z representing -0- or -NH-, and not being an integer varying from 5 to 20; arm X can be substituted by one or more groups such as -OH, -NH ₂ , -COOH, so as to contribute to the solubilization or to allow the optional derivatization of compound I; preferably the oligopeptide represented by - (P ₁ ) _x - (L-Ala-L-Ala-P ₂ ) - does not have more than 10 amino acids; it being understood that the C-terminal group represented by A can be not only a carboxylic group or one of its derivatives but also one of the groups indicated above.

Mention may in particular be made, among the carboxylic derivatives of the function which A may represent, of esters, in particular esters of formula -CO-OY, Y being an optionally substituted aliphatic, aryl or arylaliphatic group.

Y is in particular an alkyl group having 1 to 5 carbon atoms or an optionally substituted phenyl group of phenylalkyl.

Among the derivatives of the carboxylic group which A may represent, there may also be mentioned the metal salts, in particular the alkali or alkaline earth metal salts (in particular sodium, potassium, calcium, etc.), the ammonium salts, and the salts formed with amino products such as for example ethanolamine, lysine, arginine, betaines, pyridoxine (as a cofactor for lysyl oxidase involved in the synthesis of elastin), and other basic molecules, including vitamins.

A subject of the invention is also a process for the preparation of the compounds of formula I, according to the known methods for the preparation of peptides and their derivatives.

• H-X₁-(-P₁-)-(L-Ala-L-Ala-P₃-)-A₁ (II)

dans laquelle X₁ a la même définition que X ou bien représente une liaison covalente directe entre H- et -P₁-;

• P₃ a la même définition que P₂, ou P₃ représente un groupement -L-Pro-, ou bien P₃ représente une liaison covalente directe entre A₁ et le groupement -L-Ala- immédiatement adjacent;
• A_I représente un groupement - C0₂H, - CO-OY (Y étant défini comme précédemment), - CHO ou - CONH₂;
• et x est défini comme précédemment;

que l'on fait réagir ledit produit de départ, éventuellement présent sous la forme d'un sel d'addition comme le chlorhydrate, avec un réadtif de

• R-X₂-Z₁ (III)

dans laquelle R est défini comme précédemment

• X₂ a la même définition que X lorsque X₁ représente une liaison convalente, et X₂ représénte une liaison convalente directe entre R et Z₁ lorsque X₁ a la même définition que X; et Z_I est un groupement réactif permettant la réaction de R-X₂-Z₁ sur le composé II avec éliminition d'un composé Z₁H et formition d'un composé de formule IV
  • R-X-(P₁)_x-(L-Ala-L-Ala-P₃-)_y-A₁ (IV);
  
  que, dans le cas où P₃ représente le groupement -L-Pro-, on fait réagir le composé de formule IV avec un acide aminé de la série L choisi parmi la valine, l'alanine, la phénylalaline, la leucine, la méthionine et la glycine, ou avec un dérivé d'un desdits acides aminés, (en particulier un dérivé dont le groupement C-terminal est un groupement A tel que défini précédemment), pour obtenir un dérivé de formule I; puis que l'on transforme, si désiré, le composé obtenu en tout autre composé de formule 1 selon les méthodes connues, en particulier en remplaçant le groupement terminal A ou A₁ en tout autre groupement terminal répondant à la définition de A donnée ci-dessus.

The subject of the invention is in particular a process for preparing the compounds of formula 1, characterized in that a compound of general formula II is used as starting material

• HX ₁ - (- P ₁ -) - (L-Ala-L-Ala-P ₃ -) - A ₁ (II)

in which X ₁ has the same definition as X or else represents a direct covalent bond between H- and -P ₁ -;

• P ₃ has the same definition as P ₂ , or P ₃ represents a group -L-Pro-, or else P ₃ represents a direct covalent bond between A ₁ and the group -L-Ala- immediately adjacent;
• A _I represents a group - C0 ₂ H, - CO-OY (Y being defined as above), - CHO or - CONH ₂ ;
• and x is defined as above;

that said starting material is reacted, optionally present in the form of an addition salt such as the hydrochloride, with a reagent of

• RX ₂ -Z ₁ (III)

where R is defined as above

• X ₂ has the same definition as X when X ₁ represents a converse bond, and X ₂ represents a direct converse bond between R and Z ₁ when X ₁ has the same definition as X; and Z _I is a reactive group allowing the reaction of RX ₂ -Z ₁ on the compound II with elimination of a compound Z ₁ H and formation of a compound of formula IV
  • RX- (P ₁ ) _x - (L-Ala-L-Ala-P ₃ -) _y -A ₁ (IV);
  
  that, in the case where P ₃ represents the group -L-Pro-, the compound of formula IV is reacted with an amino acid of the L series chosen from valine, alanine, phenylalaline, leucine, methionine and glycine, or with a derivative of one of said amino acids, (in particular a derivative whose C-terminal group is a group A as defined above), to obtain a derivative of formula I; then transforming, if desired, the compound obtained into any other compound of formula 1 according to known methods, into in particular by replacing the terminal group A or A ₁ in any other terminal group meeting the definition of A given above.

• - Z₁ est par exemple un halogène tel que le chlore ou le brome;
• - pour préparer un composé de formule 1 pour lequel A représente -CH₂OH, on peut notamment faire réagir le composé IV (avec P₃=L-Pro) avec un dérivé d'acide aminé pour lequel le groupement C-terminal est -CH₂0H-; on opère par exemple en faisant réagir ledit dérivé (tel que le vilinol) en présence de N-méthyl morpholine et de chlorure de t-butyl carbonyle; voir notamment ANDERSON C.W. et al, J. Am. Chem. Soc., 89, 5012 (1967).
• - pour préparer un composé de formule 1 pour lequel A représente -CHO, on peut par exemple soumettre un composé de formule 1 pour lequel A représente -CH₂OH à l'action d'un agent d'oxidation tel que par exemple le diméthylsulfoxyde (PFITZNER et al, J. Am. Chem. Soc, 87, 7661 (1965) en présence d'un catalyseur approprié comme l'acide phosphorique ou dichloroacétique; THOMPSON C.R., Biochemestry, 12, 47 (1973);
• - pour préparer un composé de formule 1 dans laquelle A représente -COCH₂CI, on peut par exemple faire réagir le composé IV (P₃ = L-Pro) avec un dérivé d'acide aminé dont le groupement C-terminal est -CO-CH₂CI; voir par exemple THOMPSON et al C.R., Biochemistry, 12,44 (1973).
• - pour préparer un composé de formule 1 pour lequel A représente le groupement ester -CO-OY, on fait réagir par exemple le composé de formule 1 (A=-C0_2H) avec l'alcool choisi en présence d'un agent déshydratant tel que le chlorure de thionyle;
• - pour préparer un composé de formule 1 pour lequel A représente -CONH₂, on peut par exemple faire réagir le composé de formule IV (P₃= L-Pro) avec un acide aminé dont le groupement C-terminal est -CONH₂, par la

In preferred embodiments, the method of the invention can also have the following characteristics taken individually or in combination:

• - Z ₁ is for example a halogen such as chlorine or bromine;
• - to prepare a compound of formula 1 for which A represents -CH ₂ OH, it is possible in particular to react compound IV (with P ₃ = L-Pro) with an amino acid derivative for which the C-terminal group is - CH ₂ OH-; one operates for example by reacting said derivative (such as vilinol) in the presence of N-methyl morpholine and t-butyl carbonyl chloride; see in particular ANDERSON CW et al, J. Am. Chem. Soc., 89, 5012 (1967).
• - To prepare a compound of formula 1 for which A represents -CHO, one can for example subject a compound of formula 1 for which A represents -CH ₂ OH to the action of an oxidizing agent such as for example dimethylsulfoxide (PFITZNER et al, J. Am. Chem. Soc, 87, 7661 (1965) in the presence of a suitable catalyst such as phosphoric or dichloroacetic acid; THOMPSON CR, Biochemestry, 12, 47 (1973);
• - To prepare a compound of formula 1 in which A represents -COCH ₂ CI, it is possible, for example, to react compound IV (P ₃ = L-Pro) with an amino acid derivative whose C-terminal group is -CO -CH ₂ CI; see for example THOMPSON et al CR, Biochemistry, 12.44 (1973).
• - To prepare a compound of formula 1 for which A represents the ester group -CO-OY, the compound of formula 1 (A = -C0 _2H ) is reacted for example with the alcohol chosen in the presence of a dehydrating agent such than thionyl chloride;
• to prepare a compound of formula 1 for which A represents -CONH ₂ , it is possible, for example, to react the compound of formula IV (P ₃ = L-Pro) with an amino acid whose C-terminal group is -CONH ₂ , over there

mixed anhydrides method; Thompson et al, Biochemistry, 12, 57 (1973).

The compounds of formula 1 have interesting properties. They have in particular the dual property of inhibiting the activity of proteases of the elastase type and of binding to the elastin fiber.

In addition, they have no appreciable toxicity at the active doses. However, the compounds of formula 1 for which A represents -CO-CH ₂ Cl have a certain toxicity, generally at doses greater than 20 mg / Kg, but these compounds have a very high elastase inhibiting activity, so that their therapeutic index is no less favorable than for the other compounds of formula 1.

The subject of the present invention is also the use of the compounds of formula 1 as inhibitors of elastases and / or protectors of the elastic fiber, in particular in compositions comprising a compound of formula I with an appropriate excipient.

The compounds of formula 1 can be used, for example, as medicaments as a main treatment or as a complementary treatment in the case of arteriosclerosis, pulmonary emphysema, osteoarthritis, diabetes and certain tumors in which elastases can be involved.

The compositions of the invention are in particular pharmaceutical compositions characterized in that they comprise, as active ingredient, at least one compound of formula I, optionally in admixture with an appropriate excipient.

These pharmaceutical compositions are administered parenterally, rectally, topically or orally, or by inhalation of aerosols.

For this purpose they can be presented in the form of aqueous solutions (injectable or drinkable solutions) or solutions in pressurized packaging for aerosols of emulsions, semi-solid preparations (creams, suppositories), or in the form of lyophilized powder to be diluted or contained in an ingestible capsule or capsule.

In the pharmaceutical compositions of the invention (with the exception of lyophilized powders, the compounds of formula I are generally present at a concentration of 0.1 to 5% by weight.

The dosage depends in particular on the route of administration and the therapeutic effect sought. For example in adults it can vary from 50 mg to 5 g of active ingredient per day.

The compounds of formula I also have advantageous properties when applied to the skin, in particular properties for inhibiting cutaneous elastolysis. In particular, they make it possible to conserve or restore the suppleness of the skin and to prevent or delay the formation of wrinkles, in particular on the skin of the face, neck and hands (anti-aging effect), and the invention also has for subject use of the compounds of formula I for this purpose.

The compounds of formula 1 are therefore capable of improving the appearance of the skin and the present invention also relates to cosmetic compositions for the skin, characterized in that they comprise at least one compound of formula 1.

They also comprise at least one adjuvant or excipient usually used in cosmetic compositions for the skin.

These cosmetic compositions for the skin can be presented, for example, in the form of a cream, gel, emulsion or aqueous alcoholic or hydroalcoholic solution.

The concentration of the compound of formula I in these skin compositions generally ranges from 0.1 to 2% by weight.

The adjuvants generally present in these cosmetic compositions are, for example, perfumes, dyes, preserving agents, thickening agents or emulsifying agents.

These skin compositions in particular constitute body creams, milks or lotions, hands or face, including sunscreen creams, milks or lotions.

The subject of the invention is also a cosmetic treatment method, characterized in that at least one compound of formula 1 is applied to the skin using a cosmetic composition as defined above.

The following examples illustrate the invention without however limiting it:

Example 1 Preparation of oleoyl-L-Alanyl-L-Alanyl-L-Prolyl-L-Alanine

3.65 g (0.01M) of L-Alanyl-L-Alanyl-L-Propyl-L-Alanine (hydrochloride) prepared by a known method are dissolved in a mixture of 60 ml of 90% ethanol and 3, 35g of triethylamine. Is added thereto with stirring, at 4 ° C Ig (0.025M) of oleoyl chloride dropwise for 15 minutes and then stirred a further 4 hours at room temperature.

The ethanol is removed in vacuo, 100 ml of water are added to the residue. The pH is adjusted to 8.5 by the addition of triethylamine, the excess oleic acid is extracted with petroleum ether. The pH of the aqueous phase is adjusted to 4 by the addition of acetic acid. The oleoyl peptide is extracted with ethyl acetite.

• F = 115-120°C; a_D = -76° (C= 0,5 %, éthanol).

The solvent is removed in vacuo. The residue is dissolved in the minimum amount of ethyl acetate and the crystallization is initiated by the addition of petroleum ether.

• Mp 115-120 ° C; a _D = -76 ° (C = 0.5%, ethanol).

Example 2 Preparation of lauroyl-trialanine

1 g of Tri-Alanine is dissolved in 15 ml of 80% ethanol in the presence of 0.6 g (0.83 ml) of triethylamine. 1.1 g (1.19 ml) of lauroyl chloride are added thereto dropwise with stirring at room temperature.

The mixture is left for 2 hours at room temperature. 5 ml of water are added thereto, the mixture is extracted with petroleum ether, 30 ml of water are added to the lower phase and the mixture is left to stand for 1 hour at room temperature. It is filtered and the crystals are washed with petroleum ether and with water.

• F:219°C α_D = -86° (C = 0,5 %, éthanol).

The substance is recrystallized from 80% alcohol.

• M: 219 ° C α _D = -86 ° (C = 0.5%, ethanol).

Example 3

• Oleoyl-L-Alanyl-L-Alanyl-L-Prolyl-L-Alaninal.
• 3a) Oléoyl-L-Alanyl-L-Alanyl-L-Proline methyl ester.
• 3.5 g of oleic acid are dissolved in 1.2 ml of tetrahydrofuran.
• 1.32 ml of N-methyl morpholine and 1.50 ml of t-butyl carbonyl chloride are added to it at -15 ° C.

The mixture is stirred for 10 minutes at this temperature, then 3.08 g of L-alanyl-L-alanyl-L-proline methyl ester hydrochloride are added thereto, dissolved in a mixture of 10 ml of dimethylformamide and 1.2 ml of N-methylmorpholine.

The temperature is allowed to rise to 0 °, the mixture is stirred for 1 hour at this temperature and left to stand overnight at room temperature.

The precipitated crystals of N-methylmorpholine hydrochloride are drained.

• α_D = - 76° (C=0,5 % éthanol)
• Rf: 0,5, couche de silice, méthanol-chloroforme 5:95 v/v.
• 3b) Oléoyl-L-Alanyl-L-Alanyl-L-Proline.

The solvent is removed from the mother liquors under vacuum and the substance is crystallized from a mixture of ethyl acetate-hexane.

• α _D = - 76 ° (C = 0.5% ethanol)
• Rf: 0.5, silica layer, methanol-chloroform 5:95 v / v.
• 3b) Oleoyl-L-Alanyl-L-Alanyl-L-Proline.

• On reprend le résidu dans l'acétate d'éthyle, et on cristallise le produit dans l'éther-acétate d'éthyle.
• α_D = - 78° (C = 0,5 % éthanol).
• 3c) Oléoyl-Alanyl-Alanil-Prolyl-Alaninol.
• On dissout 1,04g (2mM) d'oléoyl-Alanyl-Alanyl-Proline dans 20ml de diméthylformamide.

1.07 g of oleoyl-L-alanyl-L-alanyl-Lproline methyl ester are dissolved in 10 ml of methanol. 3 ml of an aqueous NaOH solution (1 M) are added thereto and the solution is left for 3 hours at room temperature, the pH is adjusted to 4 using HCl, and the solvent is removed in vacuo.

• The residue is taken up in ethyl acetate, and the product is crystallized from ether-ethyl acetate.
• α _D = - 78 ° (C = 0.5% ethanol).
• 3c) Oleoyl-Alanyl-Alanil-Prolyl-Alaninol.
• 1.04 g (2 mM) of oleoyl-Alanyl-Alanyl-Proline are dissolved in 20 ml of dimethylformamide.

0.24 ml (2.2 mM) of N-methyl morpholine and 0.29 ml (2.2 mM) of t-butyl carbonyl chloride are added thereto at -15 ° C. The mixture is stirred for 10 minutes at this temperature, then 0.156 ml of L-Alaninol is added thereto. The temperature is allowed to rise to 0 ° C. and the mixture is stirred for 2 hours at this temperature. It is left to stand overnight at room temperature. The solvent is removed in vacuo, the residue is taken up in ethyl acetate, the phase is washed successively with water, with a 5% aqueous hydrochloric acid solution and with a 5% aqueous sodium carbonate solution.

• a_D = - 72° (C = 0,5%, éthanol).
• 3d) Oléoyl-Alanyl-Alanyl-Prolyl-Alaninal.

The solvent is removed in vacuo. The residue is dissolved in the minimum amount of ethyl acetate and the crystallization is initiated by the addition of petroleum ether (deliquescent crystals).

• a _D = - 72 ° (C = 0.5%, ethanol).
• 3d) Oleoyl-Alanyl-Alanyl-Prolyl-Alaninal.

2.9 g (5 mM) of oleoyl-Alanyl-Alanyl-Prolyl-Alaninol are dissolved in 18 ml of chloroform. 2 ml of dimethyl sulfoxide and 3.1 g (15 mM) of dicyclohexylcarbodiimide are added thereto.

Then added to the stirred solution to 0.86 ml (15 mM) of 90% phosphoric acid in one hour, distributed in 6 equal doses.

• α_D= -70° (C = 0,5 %, éthanol)
• R_f = 0,8, couche de silice, chloroforme-méthanol = 15:1.

After 4 hours of stirring, the solvent is removed in vacuo. The residue is taken up in 30 ml of chloroform and the solution is cooled to -30 ° C, the crystals are drained separately (dicyclohexylurea) and the mother liquors are washed with water. The substance can be isolated from the aqueous phase by preparative thin layer chromatography

• α _D = -70 ° (C = 0.5%, ethanol)
• R _f = 0.8, silica layer, chloroform-methanol = 15: 1.

Example 4 Preparation of oleoyl-tri alanine.

2 g of L-alanyl-L-alanyl-L-alanine are dissolved in 30 ml of 80% ethanol, in the presence of 1.5 g of triethylamine.

4.0 g (4.4 ml) of oleoyl chloride are added dropwise thereto.

The mixture is stirred for 2 hours at room temperature and then 50 ml of water is added thereto, followed by 1 ml of acetic acid.

The precipitate is filtered off, and washed with petroleum ether and water.

• F: 200°C; α_D = -60° (C=0,5 % éthanol).

The substance is recrystallized from alcohol.

• MP: 200 ° C; α _D = -60 ° (C = 0.5% ethanol).

Example 5 Preparation of caproyl-L-alanyl-L-alanyl-L-alanine.

1.5 g of L-alanyl-L-alanyl-L-alamine are dissolved in 20 ml of 75% ethanol containing 1.56 ml of triethylamine.

1.35 ml of caproyl chloride are added dropwise thereto, with stirring at room temperature, over 15 minutes.

The mixture is left for 2 hours at room temperature, then 30 ml of water and 2 ml of acetic acid are added thereto.

The mixture is left for 2 hours at 0 ° and then the crystals are drained and washed with 2 x 5 ml of ice water and ether.

• F:226°C; α_D = -43° (C = 0,5 % éthanol).

The substance is recrystallized from 70% alcohol.

• Mp: 226 ° C; α _D = -43 ° (C = 0.5% ethanol).

Example 6 Preparation of oleoyl-L-alanyl-L-alanyl-L-prolyl-L-valine.

0.98 g of L-alanyl-L-alanyl-L-prolyl-L-valine (hydrochloride) are dissolved in 15 ml of absolute ethanol containing 1 ml of triethylanine.

1.26 ml of oleoyl chloride are added thereto, with stirring, at 4 °, dropwise for 15 minutes, then the mixture is stirred for a further 4 hours at room temperature.

The pH is adjusted to 8.5 by addition of NaOH, and the solvent is removed in vacuo. The residue is triturated with petroleum ether and the petroleum ether is decanted.

The residue is taken up in 30 ml of water and then the pH is adjusted to 4 by addition of acetic acid; the oleoyl peptide is extracted with ethyl acetate. The solvent is removed in vacuo, and the product is crystallized from a mixture of ethyl acetate-petroleum ether.

• a_D = - 72° (C = 0,5 % éthanol).

M: 70 ° C (deliquescemt).

• a _D = - 72 ° (C = 0.5% ethanol).

• F° aD"
• Lauroyl - L-Alanyl- L-Alanyl- L-Alanine 219 -86
• Oléoyl - L-Alanyl-L-Alanyl- L-Alanine 200 (déc) -60
• Stéaroyl- L-Alanyl- L-Alanyl- L-Alanine, 212 -80
• Oléoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Valinol -70
• Oléoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Valinal -68
• * C = 0,5 %, éthanol.
• Etude des propriétés des composés de formule 1.
• 1. Expériences in vitro
• 1.a. Interaction de ces composés avec l'élastine soluble.

In analogous fashion, the following derivatives of formula 1 were prepared:

• F ° aD "
• Lauroyl - L-Alanyl- L-Alanyl- L-Alanine 219 -86
• Oleoyl - L-Alanyl-L-Alanyl- L-Alanine 200 (dec) -60
• Stearoyl- L-Alanyl- L-Alanyl- L-Alanine, 212 -80
• Oleoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Valinol -70
• Oleoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Valinal -68
• * C = 0.5%, ethanol.
• Study of the properties of the compounds of formula 1.
• 1. In vitro experiments
• 1.a. Interaction of these compounds with soluble elastin.

• 01* -(Ala)₂ Pro Ala Activité spécifique: 2.310³ cpm/nanomole
• 01* -(Ala)₂ Pro Val Activité spécifique: 2.210³ cpm/nanomole
• Remarque: "01" est une abréviation pour: oléoyl.

Soluble elastin was purified from the broad ligament of adult beef by the method using 0.1N sodium hydroxide at boiling point. Preparation of insoluble and soluble elastins. ROBERT L. and HORNEBECK W. in "The Methodology of Connective Tissue Research". Ed. DA Hall (Joynson - Bruvvers Ltd, Oxford). pp. 81-104 (1976). In order to study the adsorption of oleoylated derivatives on these polymers, the oleoylated- (Ala) ₂ -Pro-Ala and oleoyl- (Ala) ₂ -Pro-Val derivatives were synthesized using radiolabeled oleic acid.

• 01 * - (Ala) ₂ Pro Ala Specific activity: 2.310 ³ cpm / nanomole
• 01 * - (Ala) ₂ Pro Val Specific activity: 2.210 ³ cpm / nanomole
• Note: "01" is an abbreviation for: oléoyl.

Elastin (at different concentrations) and these radioactive compounds (at different concentrations), are incubated for 24 hours at 37 ° C in 1 ml of a buffer solution (100mM tris HCI, CaCl ₂ 5mM, NaN ₃ 0.02% pH 8.0).

The tubes are centrifuged at 10,000 g and the residue hydrolyzed with 1 M potassium hydroxide in the presence of 80% ethanol. The radioactivity contained in these hydrolysates is quantified and the results expressed in mmol of substance absorbed per mg of elastin.

The results obtained show that these compounds bind to elastin.

1.b. These substances and in particular alaninal derivatives act as inhibitors of elastases.

Purified elastases used: porcine pancreatic elastase (120 U / mg) and human leukocyte elastase purified from the spleen.

Molar concentration of these enzymes: Pancreatic elastase: 4.10 - ⁹ M

Leukocyte elastase: 10.10 ⁹ M

The enzymatic activity of these elastases is followed by the hydrolysis of specific synthetic substrates: N Succionoyltrialanine paranitroanilide, Acetyl-bis alanyl-Propyl-alanine paranitroanilide at a final concentration of 1.25 mM in a Tris / HC1 buffer pH 8 - 8, 6. The variation in optical density at 410nm is followed directly in a Beckman spectrophotometer type Acta C III.

In the inhibition experiments, the oleyl compounds are preincubated for 15 minutes with the elastases before determining the residual enzymatic activities.

1.c. L'élastine prétraitée avec ces substances est partiellement réfractaire à l'hydrolyse enzymatique par les élastases.The results are summarized in Table 1.

1 C. Elastin pretreated with these substances is partially refractory to enzymatic hydrolysis by elastases.

For these experiments, elastin was radiolabelled with tritiated borohydride Na B ³ H ₄ , Act sp. 1.7510 ⁵ cpm / mg. 1 mg of elastin is treated with 10 ml of a solution of the various compounds in 1 ml of Tris HCl pH 8.0 buffer for 24 hours; the mixture is centrifuged and the insoluble elastin washed with 1 ml of buffer 0.05 mg of pancreatic elastase are then added and the hydrolysis of the polymer is quantified by measuring the radioactive peptides released during the hydrolysis.

The results obtained are summarized in Table 2.

2. In vivo study of the inhibitory action of Oleoyl-bis alanyl-Proline-Alanine towards the enzymatic degradation of skin elastic fibers induced by intradermal injection of pancreatic elastase.

For these experiments, young rabbits of Garenne (1 month) were used, 3 types of intradermal injections (volume: 0.25ml; solvent: phosphate buffer pH 7.0) were performed on the backs of the experimental animals.

• 1. Control injections: 0.25m1 injection of phosphate buffer alone
• 2. Pancreatic elastase injections from pigs: 10 micrograms (1.2 units 4.10 ¹⁰ moles) in 0.25 ml of buffer.
• 3. Injections of 50-250 micrograms of Ol- (Ala) ₂ -Pro-Ala (8.10-8 ^- 3.10- ⁷ mole) in 0.25 ml of buffer followed by an injection at the same site of 10 micrograms (1.2 units 4.10 ¹⁰ mol) of pancreatic elastase in 0.25 ml of buffer.

The different rabbit skin samples are then treated in histology in order to allow the visualization of the elastic fibers.

• - que 40-60% du tissu élastique sont préservés dans le cas du prétraitement avec le dérivé oléoylé (Rapport molaire inhibiteur/enzyme = 2.10²).
• - que 60-80% du tissu élastique sont préservés dans le cas du prétraitement du tissu avec le dérivé oléoylé (Rapport molaire inhibiteur/enzyme = 103).

Histological observation and quantitative morphometry studies show:

• - that 40-60% of the elastic tissue is preserved in the case of pretreatment with the oleoylated derivative (Inhibitor / enzyme molar ratio = 2.10 ² ).
• - that 60-80% of the elastic tissue is preserved in the case of the pretreatment of the tissue with the oleoylated derivative (inhibitor / enzyme molar ratio = 103).

• a) Les substances étudiés:
  • 01-(Ala)₂-Pro-Ala,
  • 01-(Ala)₂-Pro-Val,
  • 01-(Ala)-Pro-Ala-CHO
  • agissent comme inhibiteurs des élastases;
  b) Ces composés se lient à l'élastine, et sous forme absorbée aux fibres élastiques, sont capables de diminuer considérablement l'action élastolytique des élastases, et cela aussi bien in vivo qu'in vitro.

Conclusions:

• a) The substances studied:
  • 01- (Ala) ₂ -Pro-Ala,
  • 01- (Ala) ₂ -Pro-Val,
  • 01- (Ala) -Pro-Ala-CHO
  • act as inhibitors of elastases;
  b) These compounds bind to elastin, and in absorbed form with elastic fibers, are capable of considerably reducing the elastolytic action of elastases, both in vivo and in vitro.

Claims (14)

1. Lipopeptides of general formula:

R-X-(P1)_x-(L-Ala-L-Ala-P₂) -A (I)

in which:

x denotes the number 0 or

R denotes the acyl residue of a hydrophobic carboxylic acid, such as an aliphatic carboxylic acid which has 6 to 25 carbon atoms and can optionally contain 1 to 5 double bonds, of an alicyclic carboxylic acid having 6 to 25 carbon atoms, of an arylcarboxylic acid or of an arylaliphatic carboxylic acid in which the aryl group comprises 1 or 2 rings and in which the aliphatic group comprises 1 to 18 carbon atoms, the aromatic groups of the said aryl or arylaliphatic groups optionally being substituted;

P₂ denotes an amino acid residue or dipeptide residue, connected through its N-terminal end to the adjacent L-Ala group, chosen from the group consisting of the following amino acids and dipeptides:

-L-Ala-, -L-Val-, -Gly-, -L-Met-, -L-Pro-, -L-Leu-, -L-Pro-L-Val-, -L-Pro-L-Ala-, -L-Pro-L-Phe-, -L-Pro-L-Leu, -L-Pro-L-Met- and -L-Pro-Gly-;

P₁ denotes an amino acid residue or oligopeptide residue consisting of 2 to 8 amino acids of the L- series, the said amino acids being chosen from the group consisting of glycine, alanine, valine, methionine, leucine, isoleucine, proline, phenylalanine, serine, cysteine, cystine, arginine, tyrosine, ornithine, lysine and glutamic acid,

with the proviso that P₁ is connected to RX-through its N-terminal group and that P₂ is connected to A through its C-terminal group,

Ala is the conventional representation of alanine,

X denotes a direct covalent bond connecting R either to the N-terminal group (-NH-) of the first amino acid of P₁ (in the case where x = 1), or to the N-terminal group (-NH-) of the first Ala group shown at the left in the formula I (in the case where x = 0),

or alternatively X is a divalent group having 2 to 10 carbon atoms, performing the role of "arm" between the group R and the remainder of the molecule of formula I,

and A denotes the C-terminal portion of the peptide -(P₁)_x-(Ala-Ala-P2) -A, A being chosen from a carboxylic group -C02H or the derivatives thereof (salts, esters), -CHO, -CONH₂, -COCH₂CI and -CH₂0H.

2. Lipopeptides according to Claim 1, characterized in that R denotes the acyl residue of a fatty acid having 6 to 20 carbon atoms, such as lauric acid or oleic acid, a chenodeoxycholic acid or cholic acid residue, or the acyl residue of a phenylalkanoic acid optionally substituted on the benzene ring.

3. Lipopeptides according to any one of the preceding claims, characterized in that X is a group -Z-(CH₂)_n-CO-, Z denoting -O- or -NH-, n being an integer varying from 5 to 20.

4. Lipopeptides according to any one of the preceding claims, characterized in that the oligopeptide represented by -(P1)_x-(L-Ala-L-Ala-P2) does not possess more than 10 amino acids.

5. Lipopeptides according to any one of the preceding claims characterized in that A denotes a group -CO-OY, Y being an optionally substituted arylaliphatic, aliphatic or aryl group.

6. Lipopeptides according to Claim 1, characterized in that they are chosen from the following:

- Oleoyl-L-Alanyl-L-Alanyl-L-Prolyl-L-Alanine;

- Lauroyl-trialanine;

- Oleoyl-Alanyl-Alanyl-Proline,

- Oleoyl-Alanyl-Alanyl-Prolyl-Alaninol;

- Oleoyl-Alanyl-Alanyl-Prolyl-Alaninal;

- Caproyl - L-Alanyl- L-Alanyl- L-Alanine;

- Lauroyl - L-Alanyl- L-Alanyl- L-Alanine;

- Oleoyl - L-Alanyl- L-Alanyl- L-Alanine;

- Stearoyl- L-Alanyl- L-Alanyl- L-Alanine;

- Oleoyl - L-Alanyl- L-Alanyl- L-Proline;

- Oleoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Alanine;

- Oleoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Valine;

- Oleoyl - L-Alanyl- L-alanyl- L-Prolyl- L-Alaninol;

- Oleoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Valinol;

- Oleoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Alaninol;

- Oleoyl - L-Alanyl- L-Alanyl- L-Prolyl- L-Valinal.

7. Process for preparing the lipopeptides according to any one of the preceding claims, characterized in that there is used as the starting substance a compound of general formula II

H-X₁-(-P₁-)_x-(L-Ala-L-Ala-P₃-) -A₁ (II) in which X₁ has the same definition as X or alternatively denotes a direct covalent bond between H- and -P₁-;

P3 has the same definition as P₂, or P₃ denotes an -L-Pro- group, or alternatively P₃ denotes a direct covalent bond between AI and the immediately adjacent -L-Ala- group;

A, denotes a group -C02H, -CO-OY (Y being as defined above), -CHO or -CONH₂,.

and X is defined as above;

in that the said starting substance, optionally present in the form of an addition salt such as the hydrochloride, is reacted with a reagent of formula III

R-X₂-Z₁ (III)

in which R is defined as above;

X₂ has the same definition as X when X₁ denotes a covalent bond, and X₂ denotes a direct covalent bond between R and Z₁ when X_t has the same definition as X; and Z₁ is a reactive group which enables R-X₂-Z₁ to be reacted with the compound II, with elimination of a compound Z₁H and formation of a compound of formula IV;

R-X-(P₁)_x-(L-Ala-L-Ala-P₃-) -A, (IV),

in that, in the case where P₃ denotes a -L-Progroup, the compound of formula IV is reacted with an amino acid of the L- series chosen from valine, alanine, phenylalanine, leucine or methionine and glycine, or with a derivative of one of the said amino acids, to obtain a derivative of formula I; and in that, if desired, the compound obtained is then converted to any other compound of formula I according to known methods, especially by replacing the terminal A or A₁ group with any other terminal group corresponding to the definition of A given above.

8. Use of the lipopeptides as defined in any of Claims 1 to 6 as elastase inhibitors and/or as protective agents for elastic fibres.

9. Use according to Claim 8, characterized in that the said lipopeptides are used as inhibitors of elastase in the skin and as protective agents for the skin.

10. Use according to Claim 9, characterized in that the said lipopeptides are applied on the skin for the purpose of preserving or restoring the suppleness of the skin and preventing or delaying the formation of wrinkles.

11. Compositions which are inhibitory to proteases of the elastase type and/or are protective of elastic fibres, characterized in that they contain at least one lipopeptide as defined in any one of Claims 1 to 6, mixed with a suitable excipient.

12. Compositions according to Claim 11, characterized in that they constitute a pharmaceutical composition containing the said lipopeptide as active ingredient, the said composition being usable, in particular, by way of a main or supplementary treatment of arteriosclerosis, pulmonary emphysema, osteoarthritis, diabetes and certain tumours in which elastases are implicated.

13. Compositions according to Claim 11, characterized in that they constitute a cosmetic composition containing as active ingredient at least one of the said lipopeptides, in combination with an excipient customarily used in cosmetic compositions for the skin.